Numerous microRNA (miRNA), a class of short regulatory non-coding RNAs known for their role in organ development, cellular differentiation, homeostasis, and function, have been recently demonstrated to be pivotal in regulating immune responses. We have previously shown an indispensable role of the miRNA pathway in controlling regulatory T (Treg) cell homeostasis and function. Among miRNAs highly expressed in Treg cells, miR-155 and miR-146a play pivotal roles in maintaining normal Treg cell homeostasis and more importantly regulating their suppressor function to Th1 inflammation, respectively. Nonetheless, because the complexity and the severity of the disease phenotypes in mice harboring Treg cells devoid of miRNA cannot be attributed entirely to the loss of aforementioned individual miRNAs, additional miRNAs essential for controlling other features of Treg cell biology require further elucidation. Like miR-155 and miR-146a described above, miR-23 clusters were found to be highly upregulated in Treg cells at least partially in a Foxp3 dependent manner. Moreover, the differences in the expression of miR-23 clusters between Treg cells and Tcon cells became even larger upon activation as they were further upregulated in Treg cells while downregulated in Tcon cells. Here, we propose a multifaceted study employing genetic, biochemical, immunological approaches and whole animal experimentation to comprehensively examine the molecular and cellular mechanisms underlying miR-23 cluster-mediated immune regulation. First, by generating mice harboring conditional alleles of miR-23 clusters, this loss-of-function approach will allow us to examine the role of miR-23 clusters in regulating Treg cell-mediated immunological tolerance in both physiological and autoimmune settings. Next, the immediate availability of mice harboring conditional over-expressing transgenes of the whole miR-23a cluster as well as individual miRNA member within the cluster will afford the opportunity to systematically examine the role of miR-23 cluster in controlling T cell immunity in various settings. Finally, we will explore the putative molecular mechanisms underlying miR-23 cluster-dependent immune regulation through identification of genes that are regulated in miR-23 cluster-dependent manner and through identification of targets that are directly controlled by miR-23 clusters. The proposed studies will greatly extend our fundamental knowledge of miRNA-mediated immune regulation and provide further insights into the development of strategies to manipulate Treg cell and effector T cell function as novel therapeutic approaches for treating human immunological diseases.